Negative regulation of pu.1 expression by inhibition of histone deacetylases

R. Nicholas Laribee, Michael J. Klemsz

Research output: Contribution to journalArticle


PU.l is a hematopoietic-specific member of the ETS domain family of transcription factors whose correct expression is critical for normal hematopoiesis. Deregulated expression of PU. 1 in early proerythrocytes leads to the development of murine erythroleukemia, confirming PU. 1 role as an oncogene. The ability of certain inhibitors of histone deacetylases to promote the differentiation of murine erythroleukemia cell lines prompted an examination to ascertain whether these inhibitors affected PU.l expression in cell lines that normally express the gene. Treatment of the pro-T cell line 2052 and the macrophage line P388D1 with 50nM of Trichostatin A (TSA), a histone deacetylase inhibitor, showed a specific decrease in PU.l protein expression between 8 and 15 hours. Furthermore, this decrease in PU.l expression was mirrored at the mRNA level where a significant decrease in PU.l mRNA was noted between 2 and 6 hours after TSA treatment. TSA's effect on PU. 1 expression was reversible, as cells treated for a defined period of time ( 15 hours) reexpress normal levels of PU. 1 mRNA and protein if placed into fresh media (for 24 hours) without the compound. The effect on PU. 1 expression was specific since no reduction of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) levels at the mRNA or protein levels was seen. In an attempt to understand the mechanism through which TSA exerts its negative effect on PU. 1 expression, we hypothesized that TSA may promote the acetylation of PU. 1 itself. This modification could negatively affect its ability to function as a transcriptional activator and prevent it from regulating its own expression. To address this hypothesis, we performed in vitro acetylation assays using the cloned histone acetyltransferase domains (HAT) domains from three different proteins, CREB binding protein (CBP), P300, and P300/CBP Associciated Factor (P/CAF). Each HAT domain was expressed in bacteria as a GST fusion and purified. Using as our substrate recombinantly purified histidine-tagged PU.l and GST tagged PU.l DNA binding domain, we failed to detect any acetylation of either PU.l fusion proteins. All three of our HAT domain fusions acetylated purified histones and recombinant p53. In summary, we have shown that treatment of PU. 1 expressing cells with the histone deacetylase inhibitor TSA results in a specific reduction in PU. 1 mRNA and protein levels and that this negative effect on PU. 1 expression is most likely not due to direct acetylation of PU. I. We are currently assessing whether the chromatin structure of the PU.l locus and/ or factors which regulate PU.l expression are affected by the TSA treatment.

Original languageEnglish (US)
Pages (from-to)133b
Issue number11 PART II
StatePublished - Dec 1 2000

ASJC Scopus subject areas

  • Biochemistry
  • Immunology
  • Hematology
  • Cell Biology

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